A vaccine to prevent mother-to-child-transmission of HIV-1 via breast milk, to which vulnerable infants are repeatedly exposed, is urgently needed in resource-poor countries with limited access to antiretroviral therapy. Such a vaccine requires (1) a highly accelerated vaccine schedule and (2) the rapid induction of antiviral immunity in local tissues of the oral and intestinal mucosa and their draining lymph nodes. We hypothesize that live attenuated immunogens are optimal candidates because (i) they can induce innate and adaptive immune responses against a spectrum of antigens, (ii) continue to boost these responses, and, thus, (iii) would effectively stimulate the developing infant immune system. However, for obvious safety reasons, live attenuated HIV vaccination is not feasible. Mycobacterium bovis bacilli Calmette-Guerin (BCG) vaccination induces strong T helper 1 (Th1) responses in human infants. Given the large geographical overlap between Mycobacterium tuberculosis (Mtb) and HIV infection in Africa, BCG vaccination was formerly recommended for all infants at birth to protect against tuberculosis disease (TB). However, immunodeficiency resulting from HIV-1 infection clearly predisposes infants to disseminated BCGosis. A safer TB vaccine is urgently needed. We propose to address both the urgent needs for a pediatric HIV vaccine and for a novel, safer TB vaccine by developing a highly attenuated Mtb vaccine vector (AMtb) expressing HIV antigens as a prophylactic neonatal HIV vaccine. This immunogen may protect not only against breast-milk HIV transmission, but also against Mtb infection. Both HIV-1 and TB are major causes of infant mortality and morbidity in Africa, and the successful development of a combination vaccine effective against both infections would be a major advance in the global war against pediatric diseases in developing countries. We hypothesize that highly attenuated Mtb mc25226, or its derivatives, expressing HIV antigens (rAMtb-HIV) can be safely administered orally at birth, will elicit mucosal and systemic immune responses, and thus engender protection against oral HIV infection. We will test hypothesis in the rhesus macaque model of infant oral SIV infection. AIM 1 will test the hypothesis that oral administration of mc25226- SIVgag is safe and superior to parental vaccination in eliciting local mucosal immune responses in newborn macaques. In AIM 2, we will develop strategies to optimize the immunogenicity of rAMtb in newborn macaques through genetic manipulations of immune response genes in Mtb, and define the underlying immune mechanisms by which mc25226-SIVgag enhances mucosal and systemic immune responses, and how mucosal adjuvants and various prime-boost strategies influence dendritic cell activation, CD4+T cell priming, and enhance SIV-specific CD8+T and B cell responses in infant macaques. The efficacy of a rAMtb-SIV vaccine expressing multiple SIV antigens (Gag, Pol and Env) against oral SIV challenge in infant macaques will be tested in AIM 3. Project Narrative: The goal of this project is to develop a novel oral pediatric combination vaccine that can potentially protect infants at birth against HIV infection via breast-milk transmission, and against Mycobacterium tuberculosis infection, two diseases with major impact on infant mortality and morbidity, especially in developing countries.